Methods of treatment using ctla4 molecules

ABSTRACT

The present invention provides soluble CTLA4 mutant molecules which bind with greater avidity to the CD80 and/or CD86 antigen than wild type CTLA4 or non-mutated CTLA4Ig. The soluble CTLA4 molecules have a first amino acid sequence comprising the extracellular domain of CTLA4, where certain amino acid residues within the S25-R33 region and M97-G107 region are mutated. The mutant molecules of the invention may also include a second amino acid sequence which increases the solubility of the mutant molecule.

This application is a continuation of U.S. application Ser. No.15/668,824, filed Aug. 4, 2017; which is a continuation of U.S.application Ser. No. 14/289,715, filed May 29, 2014, issued as U.S. Pat.No. 9,758,565; which is a continuation of U.S. application Ser. No.13/277,425, filed Oct. 20, 2011, issued as U.S. Pat. No. 8,785,398;which is a divisional of U.S. application Ser. No. 12/694,327, filedJan. 27, 2010, now abandoned; which is a divisional of U.S. applicationSer. No. 11/725,762, filed Mar. 20, 2007, issued as U.S. Pat. No.7,700,556; which is a divisional of U.S. application Ser. No.10/980,742, filed Nov. 3, 2004, issued as U.S. Pat. No. 7,439,230; whichis a divisional of U.S. application Ser. No. 09/865,321, filed May 23,2001, issued as U.S. Pat. No. 7,094,874; which claims priority to U.S.Provisional Application No. 60/214,065, filed Jun. 26, 2000; and U.S.Provisional Application No. 60/287,576, filed May 26, 2000. The contentsof all of the foregoing applications in their entireties areincorporated by reference into the present application.

Throughout this application various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

The present invention relates to the field of soluble CTLA4 moleculesthat are mutated from wild type CTLA4 to retain the ability to bind CD80and/or CD86.

BACKGROUND OF THE INVENTION

Antigen-nonspecific intercellular interactions between T-lymphocytes andantigen-presenting cells (APCs) generate T cell costimulatory signalsthat generate T cell responses to antigen (Jenkins and Johnson (1993)Curr. Opin. Immunol. 5:361-367). Costimulatory signals determine themagnitude of a T cell response to antigen, and whether this responseactivates or inactivates subsequent responses to antigen (Mueller et al.(1989) Annu. Rev. Immunol. 7:445-480).

T cell activation in the absence of costimulation results in an abortedor anergic T cell response (Schwartz, R. H. (1992) Cell 71:1065-1068).One key costimulatory signal is provided by interaction of the T cellsurface receptor CD28 with B7 related molecules on antigen presentingcells (e.g., also known as B7-1 and B7-2, or CD80 and CD86,respectively) (P. Linsley and J. Ledbetter (1993) Annu. Rev. Immunol.11:191-212).

The molecule now known as CD80 (B7-1) was originally described as ahuman B cell-associated activation antigen (Yokochi, T. et al. (1981) J.Immunol. 128:823-827; Freeman, G. J. et al. (1989) J. Immunol.143:2714-2722), and subsequently identified as a counterreceptor for therelated T cell molecules CD28 and CTLA4 (Linsley, P., et al. (1990)Proc. Natl. Acad. Sci. USA 87:5031-5035; Linsley, P. S. et al. (1991a)J. Exp. Med. 173:721-730; Linsley, P. S. et al. (1991b) J. Exp. Med174:561-570).

More recently, another counterreceptor for CTLA4 was identified onantigen presenting cells (Azuma, N. et al. (1993) Nature 366:76-79;Freeman (1993a) Science 262:909-911; Freeman, G. J. et al. (1993b) J.Exp. Med. 178:2185-2192; Hathcock, K. L. S., et al. (1994) J. Exp. Med.180:631-640; Lenschow, D. J. et al., (1993) Proc. Natl. Acad. Sci. USA90:11054-11058; Ravi-Wolf, Z., et al. (1993) Proc. Natl. Acad. Sci. USA90:11182-11186; Wu, Y. et al. (1993) J. Exp. Med. 178:1789-1793). Thismolecule, now known as CD86 (Caux, C., et al. (1994) J. Exp. Med.180:1841-1848), but also called B7-0 (Azuma et al., (1993), supra) orB7-2 (Freeman et al., (1993a), supra), shares about 25% sequenceidentity with CD80 in its extracellular region (Azuma et al., (1993),supra; Freeman et al., (1993a), supra, (1993b), supra). CD86-transfectedcells trigger CD28-mediated T cell responses (Azuma et al., (1993),supra; Freeman et al., (1993a), (1993b), supra).

Comparisons of expression of CD80 and CD86 have been the subject ofseveral studies (Azuma et al. (1993), supra; Hathcock et al., (1994)supra; Larsen, C. P., et al. (1994) J. Immunol. 152:5208-5219; Stack, R.M., et al., (1994) J. Immunol. 152:5723-5733). Current data indicatethat expression of CD80 and CD86 are regulated differently, and suggestthat CD86 expression tends to precede CD80 expression during an immuneresponse.

Soluble forms of CD28 and CTLA4 have been constructed by fusing variable(v)-like extracellular domains of CD28 and CTLA4 to immunoglobulin (Ig)constant domains resulting in CD28Ig and CTLA4Ig. CTLA4Ig binds bothCD80 positive and CD86 positive cells more strongly than CD28Ig(Linsley, P. et al. (1994) Immunity 1:793-80). Many T cell-dependentimmune responses are blocked by CTLA4Ig both in vitro and in vivo.(Linsley, et al., (1991b), supra; Linsley, P. S. et al., (1992a) Science257:792-795; Linsley, P. S. et al., (1992b) J. Exp. Med. 176:1595-1604;Lenschow, D. J. et al. (1992), Science 257:789-792; Tan, P. et al.,(1992) J. Exp. Med. 177:165-173; Turka, L. A., (1992) Proc. Natl. Acad.Sci. USA 89:11102-11105).

Peach et al., (J. Exp. Med. (1994) 180:2049-2058) identified regions inthe CTLA4 extracellular domain which are important for strong binding toCD80. Specifically, a hexapeptide motif (MYPPPY, SEQ ID NO:9) in thecomplementarity determining region 3 (CDR3)-like region was identifiedas fully conserved in all CD28 and CTLA4 family members. Alaninescanning mutagenesis through the MYPPPY motif (SEQ ID NO:9) in CTLA4 andat selected residues in CD28Ig reduced or abolished binding to CD80.

Chimeric molecules interchanging homologous regions of CTLA4 and CD28were also constructed. Molecules HS4, HS4-A and HS4-B were constructedby grafting CDR3-like regions of CTLA4, which also included a portioncarboxy terminally, extended to include certain nonconserved amino acidresidues onto CD28Ig. These homologue mutants showed higher bindingavidity to CD80 than did CD28Ig.

In another group of chimeric homologue mutants, the CDR1-like region ofCTLA4, which is not conserved in CD28 and is predicted to be spatiallyadjacent to the CDR3-like region, was grafted, into HS4 and HS4-A. Thesechimeric homologue mutant molecules (designated HS7 and HS8)demonstrated even greater binding avidity for CD80 than did CD28Ig.

Chimeric homologue mutant molecules were also made by grafting into HS7and HS8 the CDR2-like region of CTLA4, but this combination did notfurther improve the binding avidity for CD80. Thus, the MYPPPY motif ofCTLA4 and CD28 was determined to be critical for binding to CD80, butcertain non-conserved amino acid residues in the CDR1- and CDR3-likeregions of CTLA4 were also responsible for increased binding avidity ofCTLA4 with CD80.

CTLA4Ig was shown to effectively block CD80-associated T cellcostimulation but was not as effective at blocking CD86-associatedresponses. Soluble CTLA4 mutant molecules, especially those having ahigher avidity for CD86 than wild type CTLA4, were constructed aspossibly better able to block the priming of antigen specific activatedcells than CTLA4Ig.

There remains a need for improved CTLA4 molecules to provide betterpharmaceutical compositions for immune suppression and cancer treatmentthan previously known soluble forms of CTLA4.

SUMMARY OF INVENTION

Accordingly, the invention provides soluble CTLA4 mutant molecules thatbind CD80 and/or CD86. Mutant molecules of the invention include thosethat can recognize and bind either of CD80, CD86, or both. In someembodiments, mutant molecules bind CD80 and/or CD86 with greater aviditythan CTLA4.

One example of a CTLA4 mutant molecule is L104EA29YIg (FIG. 7), asdescribed herein. Another example of a CTLA4 mutant molecule is L104EIg(FIG. 8), as described herein. L104EA29YIg and L104EIg bind CD80 andCD86 more avidly than CTLA4Ig.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the equilibrium binding analysis of L104EA29YIg, L104EIg,and wild-type CTLA4Ig to CD86Ig.

FIGS. 2A & 2B illustrate data from FACS assays showing binding ofL104EA29YIg, L104EIg, and CTLA4Ig to human CD80- or CD86-transfected CHOcells as described in Example 2, infra.

FIGS. 3A & 3B depicts inhibition of proliferation of CD80-positive andCD86-positive CHO cells as described in Example 2, infra.

FIGS. 4A & 4B shows that L104EA29YIg is more effective than CTLA4Ig atinhibiting proliferation of primary and secondary allostimulated T cellsas described in Example 2, infra.

FIGS. 5A-C illustrate that L104EA29YIg is more effective than CTLA4Ig atinhibiting IL-2 (FIG. 5A), IL-4 (FIG. 5B), and γ-interferon (FIG. 5C)cytokine production of allostimulated human T cells as described inExample 2, infra.

FIG. 6 demonstrates that L104EA29YIg is more effective than CTLA4Ig atinhibiting proliferation of phytohemaglutinin- (PHA) stimulated monkey Tcells as described in Example 2, infra.

FIG. 7 depicts a nucleotide (SEQ ID NO: 3) and amino acid sequence (SEQID NO:4) of a CTLA4 mutant molecule (L104EA29YIg) comprising a signalpeptide; a mutated extracellular domain of CTLA4 starting at methionineat position +1 and ending at aspartic acid at position +124, or startingat alanine at position −1 and ending at aspartic acid at position +124;and an Ig region as described in Example 1, infra.

FIG. 8 depicts a nucleotide (SEQ ID NO:5) and amino acid sequence (SEQID NO:6) of a CTLA4 mutant molecule (L104EIg) comprising a signalpeptide; a mutated extracellular domain of CTLA4 starting at methionineat position +1 and ending at aspartic acid at position +124, or startingat alanine at position −1 and ending at aspartic acid at position +124;and an Ig region as described in Example 1, infra.

FIG. 9 depicts a nucleotide (SEQ ID NO:7) and amino acid sequence (SEQID NO:8) of a CTLA4Ig having a signal peptide; a wild type amino acidsequence of the extracellular domain of CTLA4 starting at methionine atposition +1 to aspartic acid at position +124, or starting at alanine atposition −1 to aspartic acid at position +124; and an Ig region.

FIGS. 10A-C are an SDS gel (FIG. 10A) for CTLA4Ig (lane 1), L104EIg(lane 2), and L104EA29YIg (lane 3A); and size exclusion chromatographsof CTLA4Ig (FIG. 10B) and L104EA29YIg (FIG. 10C).

FIG. 11A illustrates a ribbon diagram of the CTLA4 extracellular IgV-like fold generated from the solution structure determined by NMRspectroscopy. FIG. 11B shows an expanded view of the S25-R33 region andthe MYPPPY region (SEQ ID NO:9) indicating the location and side-chainorientation of the avidity enhancing mutations, L104 and A29.

FIG. 12 depicts a schematic diagram of a vector, piLN-L104EA29Y, havingthe L104EA29YIg insert.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used in this application, the following words or phrases have themeanings specified.

As used herein “wild type CTLA4” has the amino acid sequence ofnaturally occurring, full length CTLA4 (U.S. Pat. Nos. 5,434,131,5,844,095, 5,851,795), or the extracellular domain therof, which bindsCD80 and/or CD86, and/or interferes with CD80 and/or CD86 from bindingtheir ligands. In particular embodiments, the extracellular domain ofwild type CTLA4 begins with methionine at position +1 and ends ataspartic acid at position +124, or the extracellular domain of wild typeCTLA4 begins with alanine at position −1 and ends at aspartic acid atposition +124. Wild type CTLA4 is a cell surface protein, having anN-terminal extracellular domain, a transmembrane domain, and aC-terminal cytoplasmic domain. The extracellular domain binds to targetantigens, such as CD80 and CD86. In a cell, the naturally occurring,wild type CTLA4 protein is translated as an immature polypeptide, whichincludes a signal peptide at the N-terminal end. The immaturepolypeptide undergoes post-translational processing, which includescleavage and removal of the signal peptide to generate a CTLA4 cleavageproduct having a newly generated N-terminal end that differs from theN-terminal end in the immature form. One skilled in the art willappreciate that additional post-translational processing may occur,which removes one or more of the amino acids from the newly generatedN-terminal end of the CTLA4 cleavage product. The mature form of theCTLA4 molecule includes the extracellular domain of CTLA4, or anyportion thereof, which binds to CD80 and/or CD86.

“CTLA4Ig” is a soluble fusion protein comprising an extracellular domainof wild type CTLA4, or a portion thereof that binds CD80 and/or CD86,joined to an Ig tail. A particular embodiment comprises theextracellular domain of wild type CTLA4 starting at methionine atposition +1 and ending at aspartic acid at position +124; or starting atalanine at position −1 to aspartic acid at position +124; a junctionamino acid residue glutamine at position +125; and an immunoglobulinportion encompassing glutamic acid at position +126 through lysine atposition +357 (FIG. 9).

As used herein, a “fusion protein” is defined as one or more amino acidsequences joined together using methods well known in the art and asdescribed in U.S. Pat. No. 5,434,131 or 5,637,481. The joined amino acidsequences thereby form one fusion protein.

As used herein a “CTLA4 mutant molecule” is a molecule that can be fulllength CTLA4 or portions thereof (derivatives or fragments) that have amutation or multiple mutations in CTLA4 (preferably in the extracellulardomain of CTLA4) so that it is similar but no longer identical to thewild type CTLA4 molecule. CTLA4 mutant molecules bind either CD80 orCD86, or both. Mutant CTLA4 molecules may include a biologically orchemically active non-CTLA4 molecule therein or attached thereto. Themutant molecules may be soluble (i.e., circulating) or bound to asurface. CTLA4 mutant molecules can include the entire extracellulardomain of CTLA4 or portions thereof, e.g., fragments or derivatives.CTLA4 mutant molecules can be made synthetically or recombinantly.

As used herein, the term “mutation” is a change in the nucleotide oramino acid sequence of a wild-type polypeptide. In this case, it is achange in the wild type CTLA4 extracellular domain. The change can be anamino acid change which includes substitutions, deletions, additions, ortruncations. A mutant molecule can have one or more mutations. Mutationsin a nucleotide sequence may or may not result in a mutation in theamino acid sequence as is well understood in the art. In that regard,certain nucleotide codons encode the same amino acid. Examples includenucleotide codons CGU, CGG, CGC, and CGA encoding the amino acid,arginine (R); or codons GAU, and GAC encoding the amino acid, asparticacid (D). Thus, a protein can be encoded by one or more nucleic acidmolecules that differ in their specific nucleotide sequence, but stillencode protein molecules having identical sequences. The amino acidcoding sequence is as follows:

One Letter Amino Acid Symbol Symbol Codons Alanine Ala A GCU, GCC, GCA,GCG Cysteine Cys C UGU, UGC Aspartic Acid Asp D GAU, GAC Glutamic AcidGlu E GAA, GAG Phenylalanine Phe F UUU, UUC Glycine Gly G GGU, GGC, GGA,GGG Histidine His H CAU, CAC Isoleucine Ile I AUU, AUC, AUA Lysine Lys KAAA, AAG Leucine Leu L UUA, UUG, CUU, CUC, CUA, CUG Methionine Met M AUGAsparagine Asn N AAU, AAC Proline Pro P CCU, CCC, CCA, CCG Glutamine GlnQ CAA, CAG Arginine Arg R CGU, CGC, CGA, CGG, AGA, AGG Serine Ser S UCU,UCC, UCA, UCG, AGU, AGC Threonine Thr T ACU, ACC, ACA, ACG Valine Val VGUU, GUC, GUA, GUG Tryptophan Trp W UGG Tyrosine Tyr Y UAU, UAC

As used herein “the extracellular domain of CTLA4” is a portion of CTLA4that recognizes and binds CD80 and/or CD86. For example, anextracellular domain of CTLA4 comprises methionine at position +1 toaspartic acid at position +124 (FIG. 9). Alternatively, an extracellulardomain of CTLA4 comprises alanine at position −1 to aspartic acid atposition +124 (FIG. 9). The extracellular domain includes fragments orderivatives of CTLA4 that bind CD80 and/or CD86.

As used herein a “non-CTLA4 protein sequence” or “non-CTLA4 molecule” isdefined as any molecule that does not bind CD80 and/or CD86 and does notinterfere with the binding of CTLA4 to its target. An example includes,but is not limited to, an immunoglobulin (Ig) constant region or portionthereof. Preferably, the Ig constant region is a human or monkey Igconstant region, e.g., human C(gamma)1, including the hinge, CH2 and CH3regions. The Ig constant region can be mutated to reduce its effectorfunctions (U.S. Pat. Nos. 5,637,481; and 6,132,992).

As used herein a “fragment of a CTLA4 mutant molecule” is a part of aCTLA4 mutant molecule, preferably the extracellular domain of CTLA4 or apart thereof, that recognizes and binds its target, e.g., CD80 and/orCD86.

As used herein a “derivative of a CTLA4 mutant molecule” is a moleculethat shares at least 70% sequence similarity with and functions like theextracellular domain of CTLA4, i.e., it recognizes and binds CD80 and/orCD86.

As used herein, a “portion of a CTLA4 molecule” includes fragments andderivatives of a CTLA4 molecule that binds CD80 and/or CD86.

In order that the invention herein described may be more fullyunderstood, the following description is set forth.

Compositions of the Invention

The present invention provides soluble CTLA4 mutant molecules thatrecognize and bind CD80 and/or CD86. In some embodiments, the solubleCTLA4 mutants have a higher avidity to CD80 and/or CD86 than CTLA4Ig.

Examples of CTLA4 mutant molecules include L104EA29YIg (FIG. 7). Theamino acid sequence of L104EA29YIg can begin at alanine at amino acidposition −1 and end at lysine at amino acid position +357.Alternatively, the amino acid sequence of L104EA29YIg can begin atmethionine at amino acid position +1 and end at lysine at amino acidposition +357. The CTLA4 portion of L104EA29YIg encompasses methionineat amino acid position +1 through aspartic acid at amino acid position+124. L104EA29YIg comprises a junction amino acid residue glutamine atposition +125 and an immunoglobulin portion encompassing glutamic acidat position +126 through lysine at position +357 (FIG. 7). L104EA29YIgbinds approximately 2-fold more avidly than wild type CTLA4Ig(hereinafter referred to as CTLA4Ig) to CD80 and approximately 4-foldmore avidly to CD86. This stronger binding results in L104EA29YIg beingmore affective than CTLA4Ig at blocking immune responses.

CTLA4 mutant molecules comprise at least the extracellular domain ofCTLA4, or portions thereof that bind CD80 and/or CD86. The extracellularportion of a CTLA4 mutant molecule comprises an amino acid sequencestarting with methionine at position +1 through aspartic acid atposition +124 (FIG. 7 or 8). Alternatively, the extracellular portion ofthe CTLA4 can comprise an amino acid sequence starting with alanine atposition −1 through aspartic acid at position +124 (FIG. 7 or 8).

In one embodiment, the soluble CTLA4 mutant molecule is a fusion proteincomprising the extracellular domain of CTLA4 having one or moremutations in a region of an amino acid sequence beginning with serine at+25 and ending with arginine at +33 (S25-R33). For example, the alanineat position +29 of wild type CTLA4 can be substituted with tyrosine(codons: UAU, UAC). Alternatively, alanine can be substituted withleucine (codons: UUA, UUG, CUU, CUC, CUA, CUG), phenylalanine (codons:UUU, UUC), tryptophan (codon: UGG), or threonine (codons: ACU, ACC, ACA,ACG). As persons skilled in the art will readily understand, the uracil(U) nucleotide of the RNA sequence corresponds to the thymine (T)nucleotide of the DNA sequence.

In another embodiment, the soluble CTLA4 mutant molecule is a fusionprotein comprising the extracellular domain of CTLA4 having one or moremutations in or near a region of an amino acid sequence beginning withmethionine at +97 and ending with glycine at +107 (M97-G107). Forexample, leucine at position +104 of wild type CTLA4 can be substitutedwith glutamic acid (codons: GAA, GAG). A CTLA4 mutant molecule havingthis substitution is referred to herein as L104EIg (FIG. 8).

In yet another embodiment, the soluble CTLA4 mutant molecule is a fusionprotein comprising the extracellular domain of CTLA4 having one or moremutations in the S25-R33 and M97-G107 regions. For example, in oneembodiment, a CTLA4 mutant molecule comprises tyrosine at position +29instead of alanine; and glutamic acid at position +104 instead ofleucine. A CTLA4 mutant molecule having these substitutions is referredto herein as L104EA29YIg (FIG. 7). The nucleic acid molecule thatencodes L104EA29YIg is contained in pD16 L104EA29YIg and was depositedon Jun. 20, 2000 with the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209 (ATCC No. PTA-2104). The pD16L104EA29YIg vector is a derivative of the pcDNA3 vector (INVITROGEN).

The invention further provides a soluble CTLA4 mutant moleculecomprising an extracellular domain of a CTLA4 mutant as shown in FIG. 7or 8, or portion(s) thereof, and a moiety that alters the solubility,affinity and/or valency of the CTLA4 mutant molecule.

In accordance with a practice of the invention, the moiety can be animmunoglobulin constant region or portion thereof. For in vivo use, itis preferred that the immunoglobulin constant region does not elicit adetrimental immune response in the subject. For example, in clinicalprotocols, it may be preferred that mutant molecules include human ormonkey immunoglobulin constant regions. One example of a suitableimmunoglobulin region is human C(gamma)1, comprising the hinge, CH2, andCH3 regions. Other isotypes are possible. Further, other immunoglobulinconstant regions are possible (preferably other weakly ornon-immunogenic immunoglobulin constant regions).

Other moieties include polypeptide tags. Examples of suitable tagsinclude but are not limited to the p97 molecule, env gp120 molecule, E7molecule, and ova molecule (Dash, B., et al. (1994) J. Gen. Virol.75:1389-97; Ikeda, T., et al. (1994) Gene 138:193-6; Falk, K., et al.(1993) Cell. Immunol. 150:447-52; Fujisaka, K. et al. (1994) Virology204:789-93). Other molecules for use as tags are possible (Gerard, C. etal. (1994) Neuroscience 62:721-739; Byrn, R. et al. J. Virol. (1989)63:4370-4375; Smith, D. et al., (1987) Science 238:1704-1707; Lasky, L.,(1996) Science 233:209-212).

The invention further provides soluble mutant CTLA4Ig fusion proteinspreferentially more reactive with the CD80 and/or CD86 antigen comparedto wild type CTLA4. One example is L104EA29YIg as shown in FIG. 7.

In another embodiment, the soluble CTLA4 mutant molecule includes ajunction amino acid residue, which is located between the CTLA4 portionand the immunoglobulin portion. The junction amino acid can be any aminoacid, including glutamine. The junction amino acid can be introduced bymolecular or chemical synthesis methods known in the art.

In another embodiment, the soluble CTLA4 mutant molecule includes theimmunoglobulin portion (e.g., hinge, CH2 and CH3 domains), where any orall of the cysteine residues, within the hinge domain of theimmunoglobulin portion are substituted with serine, for example, thecysteines at positions +130, +136, or +139 (FIG. 7 or 8). The mutantmolecule may also include the proline at position +148 substituted witha serine, as shown in FIG. 7 or 8.

The soluble CTLA4 mutant molecule can include a signal peptide sequencelinked to the N-terminal end of the extracellular domain of the CTLA4portion of the mutant molecule. The signal peptide can be any sequencethat will permit secretion of the mutant molecule, including the signalpeptide from oncostatin M (Malik, et al., (1989) Molec. Cell. Biol. 9:2847-2853), or CD5 (Jones, N. H. et al., (1986) Nature 323:346-349), orthe signal peptide from any extracellular protein.

The mutant molecule can include the oncostatin M signal peptide linkedat the N-terminal end of the extracellular domain of CTLA4, and thehuman immunoglobulin molecule (e.g., hinge, CH2 and CH3) linked to theC-terminal end of the extracellular domain of CTLA4. This moleculeincludes the oncostatin M signal peptide encompassing an amino acidsequence having methionine at position −26 through alanine at position−1, the CTLA4 portion encompassing an amino acid sequence havingmethionine at position +1 through aspartic acid at position +124, ajunction amino acid residue glutamine at position +125, and theimmunoglobulin portion encompassing an amino acid sequence havingglutamic acid at position +126 through lysine at position +357.

The soluble CTLA4 mutant molecules of the invention can be obtained bymolecular or chemical synthesis methods. The molecular methods mayinclude the following steps: introducing a suitable host cell with anucleic acid molecule that expresses and encodes the soluble CTLA4mutant molecule; culturing the host cell so introduced under conditionsthat permit the host cell to express the mutant molecules; and isolatingthe expressed mutant molecules. The signal peptide portion of the mutantmolecule permits the protein molecules to be expressed on the cellsurface and to be secreted by the host cell. The translated mutantmolecules can undergo post-translational modification, involvingcleavage of the signal peptide to produce a mature protein having theCTLA4 and the immunoglobulin portions. The cleavage may occur after thealanine at position −1, resulting in a mature mutant molecule havingmethionine at position +1 as the first amino acid (FIG. 7 or 8).Alternatively, the cleavage may occur after the methionine at position−2, resulting in a mature mutant molecule having alanine at position −1as the first amino acid.

A preferred embodiment is a soluble CTLA4 mutant molecule having theextracellular domain of human CTLA4 linked to all or a portion of ahuman immunoglobulin molecule (e.g., hinge, CH2 and CH3). This preferredmolecule includes the CTLA4 portion of the soluble molecule encompassingan amino acid sequence having methionine at position +1 through asparticacid at position +124, a junction amino acid residue glutamine atposition +125, and the immunoglobulin portion encompassing glutamic acidat position +126 through lysine at position +357. The portion having theextracellular domain of CTLA4 is mutated so that alanine at position +29is substituted with tyrosine and leucine at position +104 is substitutedwith glutamic acid. The immunoglobulin portion of the mutant moleculecan be mutated, so that the cysteines at positions +130, +136, and +139are substituted with serine, and the proline at position +148 issubstituted with serine. This mutant molecule is designated herein asL104EA29YIg (FIG. 7).

Another embodiment of L104EA29YIg is a mutant molecule having an aminoacid sequence having alanine at position −1 through aspartic acid atposition +124, a junction amino acid residue glutamine at position +125,and the immunoglobulin portion encompassing glutamic acid at position+126 (e.g., +126 through lysine at position +357). The portion havingthe extracellular domain of CTLA4 is mutated so that alanine at position+29 is replaced with tyrosine; and leucine at position +104 is replacedwith glutamic acid. The immunoglobulin portion of the mutant molecule ismutated so that the cysteines at positions +130, +136, and +139 arereplaced with serine, and the proline at position +148 is replaced withserine. This mutant molecule is designated herein as L104EA29YIg (FIG.7). After the signal sequence has been cleaved, L104EA29YIg can eitherbegin with a methionine at position +1, or begin with alanine atposition −1.

Another mutant molecule of the invention is a soluble CTLA4 mutantmolecule having the extracellular domain of human CTLA4 linked to thehuman immunoglobulin molecule (e.g., hinge, CH2 and CH3). This moleculeincludes the portion of the amino acid sequence encoding CTLA4 startingwith methionine at position +1 through aspartic acid at position +124, ajunction amino acid residue glutamine at position +125, and theimmunoglobulin portion encompassing an amino acid sequence havingglutamic acid at position +126 through lysine at position +357. Theportion having the extracellular domain of CTLA4 is mutated so thatleucine at position +104 is substituted with glutamic acid. The hingeportion of the mutant molecule is mutated so that the cysteines atpositions +130, +136, and +139 are substituted with serine, and theproline at position +148 is substituted with serine. This mutantmolecule is designated herein as L104EIg (FIG. 8).

Alternatively, an embodiment of L104EIg is a soluble CTLA4 mutantmolecule having an extracellular domain of human CTLA4 linked to a humanimmunoglobulin molecule (e.g., hinge, CH2 and CH3). This preferredmolecule includes the CTLA4 portion encompassing an amino acid sequencebeginning with alanine at position −1 through aspartic acid at position+124, a junction amino acid residue glutamine at position +125, and theimmunoglobulin portion encompassing glutamic acid at position +126through lysine at position +357. The portion having the extracellulardomain of CTLA4 is mutated so that leucine at position +104 issubstituted with glutamic acid. The hinge portion of the mutant moleculeis mutated so that the cysteines at positions +130, +136, and +139 aresubstituted with serine, and the proline at position +148 is substitutedwith serine. This mutant molecule is designated herein as L104EIg (FIG.8).

Further, the invention provides a soluble CTLA4 mutant molecule having:(a) a first amino acid sequence of a membrane glycoprotein, e.g., CD28,CD86, CD80, CD40, and gp39, which blocks T cell proliferation, fused toa second amino acid sequence; (b) the second amino acid sequence being afragment of the extracellular domain of mutant CTLA4 which blocks T cellproliferation, such as, for example an amino acid molecule comprisingmethionine at position +1 through aspartic acid at position +124 (FIG. 7or 8); and (c) a third amino acid sequence which acts as anidentification tag or enhances solubility of the molecule. For example,the third amino acid sequence can consist essentially of amino acidresidues of the hinge, CH2 and CH3 regions of a non-immunogenicimmunoglobulin molecule. Examples of suitable immunoglobulin moleculesinclude, but are not limited to, human or monkey immunoglobulin, e.g.,C(gamma)1. Other isotypes are also possible.

The invention further provides nucleic acid molecules comprisingnucleotide sequences encoding the amino acid sequences corresponding tothe soluble CTLA4 mutant molecules of the invention. In one embodiment,the nucleic acid molecule is a DNA (e.g., cDNA) or a hybrid thereof.Alternatively, the nucleic acid molecules are RNA or a hybrids thereof.

Additionally, the invention provides a vector, which comprises thenucleotide sequences of the invention. A host vector system is alsoprovided. The host vector system comprises the vector of the inventionin a suitable host cell. Examples of suitable host cells include, butare not limited to, prokaryotic and eukaryotic cells.

The invention includes pharmaceutical compositions for use in thetreatment of immune system diseases comprising pharmaceuticallyeffective amounts of soluble CTLA4 mutant molecules. In certainembodiments, the immune system diseases are mediated by CD28- and/orCTLA4-positive cell interactions with CD80 and/or CD86 positive cells.The soluble CTLA4 mutant molecules are preferably CTLA4 molecules havingone or more mutations in the extracellular domain of CTLA4. Thepharmaceutical composition can include soluble CTLA4 mutant proteinmolecules and/or nucleic acid molecules, and/or vectors encoding themolecules. In preferred embodiments, the soluble CTLA4 mutant moleculeshave the amino acid sequence of the extracellular domain of CTLA4 asshown in either FIG. 7 or 8 (L104EA29Y or L104E, respectively). Evenmore preferably, the soluble CTLA4 mutant molecule is L104EA29YIg asdisclosed herein. The compositions may additionally include othertherapeutic agents, including, but not limited to, drug toxins, enzymes,antibodies, or conjugates.

The pharmaceutical compositions also preferably include suitablecarriers and adjuvants which include any material which when combinedwith the molecule of the invention (e.g., a soluble CTLA4 mutantmolecule, such as, L104EA29Y or L104E) retains the molecule's activityand is non-reactive with the subject's immune system. Examples ofsuitable carriers and adjuvants include, but are not limited to, humanserum albumin; ion exchangers; alumina; lecithin; buffer substances,such as phosphates; glycine; sorbic acid; potassium sorbate; and saltsor electrolytes, such as protamine sulfate. Other examples include anyof the standard pharmaceutical carriers such as a phosphate bufferedsaline solution; water; emulsions, such as oil/water emulsion; andvarious types of wetting agents. Other carriers may also include sterilesolutions; tablets, including coated tablets and capsules. Typicallysuch carriers contain excipients such as starch, milk, sugar, certaintypes of clay, gelatin, stearic acid or salts thereof, magnesium orcalcium stearate, talc, vegetable fats or oils, gums, glycols, or otherknown excipients. Such carriers may also include flavor and coloradditives or other ingredients. Compositions comprising such carriersare formulated by well known conventional methods. Such compositions mayalso be formulated within various lipid compositions, such as, forexample, liposomes as well as in various polymeric compositions, such aspolymer microspheres.

The pharmaceutical compositions of the invention can be administeredusing conventional modes of administration including, but not limitedto, intravenous (i.v.) administration, intraperitoneal (i.p.)administration, intramuscular (i.m.) administration, subcutaneousadministration, oral administration, administration as a suppository, oras a topical contact, or the implantation of a slow-release device suchas a miniosmotic pump, to the subject.

The pharmaceutical compositions of the invention may be in a variety ofdosage forms, which include, but are not limited to, liquid solutions orsuspensions, tablets, pills, powders, suppositories, polymericmicrocapsules or microvesicles, liposomes, and injectable or infusiblesolutions. The preferred form depends upon the mode of administrationand the therapeutic application.

The most effective mode of administration and dosage regimen for thecompositions of this invention depends upon the severity and course ofthe disease, the patient's health and response to treatment and thejudgment of the treating physician. Accordingly, the dosages of thecompositions should be titrated to the individual patient.

The soluble CTLA4 mutant molecules may be administered to a subject inan amount and for a time (e.g. length of time and/or multiple times)sufficient to block endogenous B7 (e.g., CD80 and/or CD86) moleculesfrom binding their respective ligands, in the subject. Blockage ofendogenous B7/ligand binding thereby inhibits interactions betweenB7-positive cells (e.g., CD80- and/or CD86-positive cells) with CD28-and/or CTLA4-positive cells. Dosage of a therapeutic agent is dependantupon many factors including, but not limited to, the type of tissueaffected, the type of autoimmune disease being treated, the severity ofthe disease, a subject's health, and a subject's response to thetreatment with the agents. Accordingly, dosages of the agents can varydepending on the subject and the mode of administration. The solubleCTLA4 mutant molecules may be administered in an amount between 0.1 to20.0 mg/kg weight of the patient/day, preferably between 0.5 to 10.0mg/kg/day. Administration of the pharmaceutical compositions of theinvention can be performed over various times. In one embodiment, thepharmaceutical composition of the invention can be administered for oneor more hours. In addition, the administration can be repeated dependingon the severity of the disease as well as other factors as understood inthe art.

The invention further provides methods for producing a proteincomprising growing the host vector system of the invention so as toproduce the protein in the host and recovering the protein so produced.

Additionally, the invention provides methods for regulating functionalCTLA4- and CD28-positive T cell interactions with CD80- and/orCD86-positive cells. The methods comprise contacting the CD80- and/orCD86-positive cells with a soluble CTLA4 mutant molecule of theinvention so as to form mutant CTLA4/CD80 and/or mutant CTLA4/CD86complexes, the complexes interfering with reaction of endogenous CTLA4antigen with CD80 and/or CD86, and/or the complexes interfering withreaction of endogenous CD28 antigen with CD80 and/or CD86. In oneembodiment of the invention, the soluble CTLA4 mutant molecule is afusion protein that contains at least a portion of the extracellulardomain of mutant CTLA4. In another embodiment, the soluble CTLA4 mutantmolecule comprises: a first amino acid sequence including theextracellular domain of CTLA4 from the amino acid sequence havingmethionine at position +1 to aspartic acid at position +124, includingat least one mutation; and a second amino acid sequence including thehinge, CH2, and CH3 regions of the human immunoglobulin gamma 1 molecule(FIG. 7 or 8).

In accordance with the practice of the invention, the CD80- orCD86-positive cells are contacted with fragments or derivatives of thesoluble CTLA4 mutant molecules of the invention. Alternatively, thesoluble CTLA4 mutant molecule is a CD28Ig/CTLA4Ig fusion protein havinga first amino acid sequence corresponding to a portion of theextracellular domain of CD28 receptor fused to a second amino acidsequence corresponding to a portion of the extracellular domain of CTLA4mutant receptor and a third amino acid sequence corresponding to thehinge, CH2 and CH3 regions of human immunoglobulin C-gamma-1.

The soluble CTLA4 mutant molecules are expected to exhibit inhibitoryproperties in vivo. Under conditions where T cell/APC cell interactions,for example T cell/B cell interactions, are occurring as a result ofcontact between T cells and APC cells, binding of introduced CTLA4mutant molecules to react to CD80- and/or CD86-positive cells, forexample B cells, may interfere, i.e., inhibit, the T cell/APC cellinteractions resulting in regulation of immune responses.

The invention provides methods for downregulating immune responses. Downregulation of an immune response by soluble CTLA4 mutant molecules maybe by way of inhibiting or blocking an immune response already inprogress or may involve preventing the induction of an immune response.The soluble CTLA4 molecules of the invention may inhibit the functionsof activated T cells, such as T lymphocyte proliferation and cytokinesecretion, by suppressing T cell responses or by inducing specifictolerance in T cells, or both.

The present invention further provides methods for treating immunesystem diseases and tolerance induction In particular embodiments, theimmune system diseases are mediated by CD28- and/or CTLA4-positive cellinteractions with CD80/CD86-positive cells. In a further embodiment, Tcell interactions are inhibited. Immune system diseases include, but arenot limited to, autoimmune diseases, immunoproliferative diseases, andgraft-related disorders. These methods comprise administering to asubject the soluble CTLA4 mutant molecules of the invention to regulateT cell interactions with the CD80- and/or CD86-positive cells.Alternatively, a CTLA4 mutant hybrid having a membrane glycoproteinjoined to a CTLA4 mutant molecule can be administered. Examples ofgraft-related diseases include graft versus host disease (GVHD) (e.g.,such as may result from bone marrow transplantation, or in the inductionof tolerance), immune disorders associated with graft transplantationrejection, chronic rejection, and tissue or cell allo- or xenografts,including solid organs, skin, islets, muscles, hepatocytes, neurons.Examples of immunoproliferative diseases include, but are not limitedto, psoriasis; T cell lymphoma; T cell acute lymphoblastic leukemia;testicular angiocentric T cell lymphoma; benign lymphocytic angiitis;and autoimmune diseases such as lupus (e.g., lupus erythematosus, lupusnephritis), Hashimoto's thyroiditis, primary myxedema, Graves' disease,pernicious anemia, autoimmune atrophic gastritis, Addison's disease,diabetes (e.g. insulin dependent diabetes mellitis, type I diabetesmellitis), good pasture's syndrome, myasthenia gravis, pemphigus,Crohn's disease, sympathetic ophthalmia, autoimmune uveitis, multiplesclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenia,primary biliary cirrhosis, chronic action hepatitis, ulceratis colitis,Sjogren's syndrome, rheumatic diseases (e.g., rheumatoid arthritis),polymyositis, scleroderma, and mixed connective tissue disease.

The present invention further provides a method for inhibiting solidorgan and/or tissue transplant rejections by a subject, the subjectbeing a recipient of transplant tissue. Typically, in tissuetransplants, rejection of the graft is initiated through its recognitionas foreign by T cells, followed by an immune response that destroys thegraft. The soluble CTLA4 mutant molecules of this invention, byinhibiting T lymphocyte proliferation and/or cytokine secretion, mayresult in reduced tissue destruction and induction of antigen-specific Tcell unresponsiveness may result in long-term graft acceptance withoutthe need for generalized immunosuppression. Furthermore, the solubleCTLA4 mutant molecules of the invention can be administered with otherpharmaceuticals including, but not limited to, corticosteroids,cyclosporine, rapamycin, mycophenolate mofetil, azathioprine,tacrolimus, basiliximab, and/or other biologics.

The present invention also provides methods for inhibiting graft versushost disease in a subject. This method comprises administering to thesubject a soluble CTLA4 mutant molecule of the invention, alone ortogether, with further additional ligands, reactive with IL-2, IL-4, or7-interferon. For example, a soluble CTLA mutant molecule of thisinvention may be administered to a bone marrow transplant recipient toinhibit the alloreactivity of donor T cells. Alternatively, donor Tcells within a bone marrow graft may be tolerized to a recipient'salloantigens ex vivo prior to transplantation.

Inhibition of T cell responses by soluble CTLA4 mutant molecules mayalso be useful for treating autoimmune disorders. Many autoimmunedisorders result from inappropriate activation of T cells that arereactive against autoantigens, and which promote the production ofcytokines and autoantibodies that are involved in the pathology of thedisease. Administration of a soluble CTLA4 mutant molecule in a subjectsuffering from or susceptible to an autoimmune disorder may prevent theactivation of autoreactive T cells and may reduce or eliminate diseasesymptoms. This method may also comprise administering to the subject asoluble CTLA4 mutant molecule of the invention, alone or together, withfurther additional ligands, reactive with IL-2, IL-4, or γ-interferon.

The invention further encompasses the use of the soluble CTLA4 mutantmolecules together with other immunosuppressants, e.g., cyclosporin (seeMathiesen, in: “Prolonged Survival and Vascularization of XenograftedHuman Glioblastoma Cells in the Central Nervous System of CyclosporinA-Treated Rats” (1989) Cancer Lett., 44:151-156), prednisone,azathioprine, and methotrexate (R. Handschumacher “Chapter 53: DrugsUsed for Immunosuppression” pages 1264-1276). Other immunosuppressantsare possible. For example, for the treatment of rheumatoid arthritis,soluble CTLA4 mutant molecules can be administered with pharmaceuticalsincluding, but not limited to, corticosteroids, nonsteroidalantiinflammatory drugs/Cox-2 inhibitors, methotrexate,hydroxychloroquine, sulphasalazopryine, gold salts, etanercept,infliximab, anakinra, azathioprine, and/or other biologics likeanti-TNF. For the treatment of systemic lupus eryhtemathosus, solubleCTLA4 mutant molecules can be administered with pharmaceuticalsincluding, but not limited to, corticosteroids, cytoxan, azathioprine,hydroxychloroquine, mycophenolate mofetil, and/or other biologics.Further, for the treatment of multiple sclerosis, soluble CTLA4 mutantmolecules can be administered with pharmaceuticals including, but notlimited to, corticosteroids, interferon beta-1a, interferon beta-1b,glatiramer acetate, mitoxantrone hydrochloride, and/or other biologics.

The soluble CTLA4 mutant molecules (preferably, L104EA29YIg) can also beused in combination with one or more of the following agents to regulatean immune response: soluble gp39 (also known as CD40 ligand (CD40L),CD154, T-BAM, TRAP), soluble CD29, soluble CD40, soluble CD80, solubleCD86, soluble CD28, soluble CD56, soluble Thy-1, soluble CD3, solubleTCR, soluble VLA-4, soluble VCAM-1, soluble LECAM-1, soluble ELAM-1,soluble CD44, antibodies reactive with gp39, antibodies reactive withCD40, antibodies reactive with B7, antibodies reactive with CD28,antibodies reactive with LFA-1, antibodies reactive with LFA-2,antibodies reactive with IL-2, antibodies reactive with IL-12,antibodies reactive with IFN-gamma, antibodies reactive with CD2,antibodies reactive with CD48, antibodies reactive with any ICAM (e.g.,ICAM-2), antibodies reactive with CTLA4, antibodies reactive with Thy-1,antibodies reactive with CD56, antibodies reactive with CD3, antibodiesreactive with CD29, antibodies reactive with TCR, antibodies reactivewith VLA-4, antibodies reactive with VCAM-1, antibodies reactive withLECAM-1, antibodies reactive with ELAM-1, antibodies reactive with CD44.In certain embodiments, monoclonal antibodies are preferred. In otherembodiments, antibody fragments are preferred. As persons skilled in theart will readily understand, the combination can include the solubleCTLA4 mutant molecules of the invention and one other immunosuppressiveagent, the soluble CTLA4 mutant molecules with two otherimmunosuppressive agents, the soluble CTLA4 mutant molecules with threeother immunosuppressive agents, etc. The determination of the optimalcombination and dosages can be determined and optimized using methodswell known in the art.

Some specific combinations include the following: L104EA29YIg and CD80mAbs; L104EA29YIg and CD86 mAbs; L104EA29YIg, CD80 mAbs, and CD86 mAbs;L104EA29YIg and gp39 mAbs; L104EA29YIg and CD40 mAbs; L104EA29YIg andCD28 mAbs; L104EA29YIg, CD80 and CD86 mAbs, and gp39 mAbs; L104EA29YIg,CD80 and CD86 mAbs and CD40 mAbs; and L104EA29YIg, anti-LFA1 mAb, andanti-gp39 mAb. A specific example of a gp39 mAb is MR1. Othercombinations will be readily appreciated and understood by personsskilled in the art.

The soluble CTLA4 mutant molecules of the invention, for exampleL104EA29Y, may be administered as the sole active ingredient or togetherwith other drugs in immunomodulating regimens or other anti-inflammatoryagents e.g. for the treatment or prevention of allo- or xenograft acuteor chronic rejection or inflammatory or autoimmune disorders, or toinduce tolerance. For example, it may be used in combination with acalcineurin inhibitor, e.g. cyclosporin A or FK506; an immunosuppressivemacrolide, e.g. rapamycine or a derivative thereof; e.g.40-O-(2-hydroxy)ethyl-rapamycin, a lymphocyte homing agent, e.g. FTY720or an analog thereof; corticosteroids; cyclophosphamide; azathioprene;methotrexate; leflunomide or an analog thereof; mizoribine; mycophenolicacid; mycophenolate mofetil; 15-deoxyspergualine or an analog thereof;immunosuppressive monoclonal antibodies, e.g., monoclonal antibodies toleukocyte receptors, e.g., MHC, CD2, CD3, CD4, CD 11a/CD18, CD7, CD25,CD 27, B7, CD40, CD45, CD58, CD 137, ICOS, CD150 (SLAM), OX40, 4-1BB ortheir ligands; or other immunomodulatory compounds, e.g. CTLA4/CD28-Ig,or other adhesion molecule inhibitors, e.g. mAbs or low molecular weightinhibitors including LFA-1 antagonists, Selectin antagonists and VLA-4antagonists. The compound is particularly useful in combination with acompound which interferes with CD40 and its ligand, e.g. antibodies toCD40 and antibodies to CD40-L, e.g. in the above described indications,e.g the induction of tolerance.

Where the soluble CTLA4 mutant molecules of the invention areadministered in conjunction with otherimmunosuppressive/immunomodulatory or anti-inflammatory therapy, e.g ashereinabove specified, dosages of the co-administered immunosuppressant,immunomodulatory or anti-inflammatory compound will of course varydepending on the type of co-drug employed, e.g. whether it is a steroidor a cyclosporine, on the specific drug employed, on the condition beingtreated and so forth.

In accordance with the foregoing the present invention provides in a yetfurther aspect methods as defined above comprising co-administration,e.g. concomitantly or in sequence, of a therapeutically effective amountof soluble CTLA4 mutant molecules of the invention, L104EA29YIg, in freeform or in pharmaceutically acceptable salt form, and a second drugsubstance, said second drug substance being an immunosuppressant,immunomodulatory or anti-inflammatory drug, e.g. as indicated above.Further provided are therapeutic combinations, e.g. a kit, e.g. for usein any method as defined above, comprising a L104EA29YIg, in free formor in pharmaceutically acceptable salt form, to be used concomitantly orin sequence with at least one pharmaceutical composition comprising animmunosuppressant, immunomodulatory or anti-inflammatory drug. The kitmay comprise instructions for its administration.

Methods for Producing the Molecules of the Invention

Expression of CTLA4 mutant molecules can be in prokaryotic cells.Prokaryotes most frequently are represented by various strains ofbacteria. The bacteria may be a gram positive or a gram negative.Typically, gram-negative bacteria such as E. coli are preferred. Othermicrobial strains may also be used.

Sequences encoding CTLA4 mutant molecules can be inserted into a vectordesigned for expressing foreign sequences in prokaryotic cells such asE. coli. These vectors can include commonly used prokaryotic controlsequences which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding site sequences, include such commonly used promoters asthe beta-lactamase (penicillinase) and lactose (lac) promoter systems(Chang, et al., (1977) Nature 198:1056), the tryptophan (trp) promotersystem (Goeddel, et al., (1980) Nucleic Acids Res. 8:4057) and thelambda derived P_(L) promoter and N-gene ribosome binding site(Shimatake, et al., (1981) Nature 292:128).

Such expression vectors will also include origins of replication andselectable markers, such as a beta-lactamase or neomycinphosphotransferase gene conferring resistance to antibiotics, so thatthe vectors can replicate in bacteria and cells carrying the plasmidscan be selected for when grown in the presence of antibiotics, such asampicillin or kanamycin.

The expression plasmid can be introduced into prokaryotic cells via avariety of standard methods, including but not limited to CaCl₂)-shock(Cohen, (1972) Proc. Natl. Acad. Sci. USA 69:2110, and Sambrook et al.(eds.), “Molecular Cloning: A Laboratory Manual”, 2nd Edition, ColdSpring Harbor Press, (1989)) and electroporation.

In accordance with the practice of the invention, eukaryotic cells arealso suitable host cells. Examples of eukaryotic cells include anyanimal cell, whether primary or immortalized, yeast (e.g., Saccharomycescerevisiae, Schizosaccharomyces pombe, and Pichia pastoris), and plantcells. Myeloma, COS and CHO cells are examples of animal cells that maybe used as hosts. Particular CHO cells include, but are not limited to,DG44 (Chasin, et la., 1986 Som. Cell. Molec. Genet. 12:555-556; Kolkekar1997 Biochemistry 36:10901-10909), CHO-K1 (ATCC No. CCL-61), CHO-K1Tet-On cell line (Clontech), CHO designated ECACC 85050302 (CAMR,Salisbury, Wiltshire, UK), CHO clone 13 (GEIMG, Genova, IT), CHO clone B(GEIMG, Genova, IT), CHO-K1/SF designated ECACC 93061607 (CAMR,Salisbury, Wiltshire, UK), and RR-CHO-K1 designated ECACC 92052129(CAMR, Salisbury, Wiltshire, UK). Exemplary plant cells include tobacco(whole plants, cell culture, or callus), corn, soybean, and rice cells.Corn, soybean, and rice seeds are also acceptable.

Nucleic acid sequences encoding the CTLA4 mutant molecules can also beinserted into a vector designed for expressing foreign sequences in aeukaryotic host. The regulatory elements of the vector can varyaccording to the particular eukaryotic host.

Commonly used eukaryotic control sequences for use in expression vectorsinclude promoters and control sequences compatible with mammalian cellssuch as, for example, CMV promoter (CDM8 vector) and avian sarcoma virus(ASV) (πLN vector). Other commonly used promoters include the early andlate promoters from Simian Virus 40 (SV40) (Fiers, et al., (1973) Nature273:113), or other viral promoters such as those derived from polyoma,Adenovirus 2, and bovine papilloma virus. An inducible promoter, such ashMTII (Karin, et al., (1982) Nature 299:797-802) may also be used.

Vectors for expressing CTLA4 mutant molecules in eukaryotes may alsocarry sequences called enhancer regions. These are important inoptimizing gene expression and are found either upstream or downstreamof the promoter region.

Examples of expression vectors for eukaryotic host cells include, butare not limited to, vectors for mammalian host cells (e.g., BPV-1, pHyg,pRSV, pSV2, pTK2 (Maniatis); pIRES (Clontech); pRc/CMV2, pRc/RSV, pSFV1(Life Technologies); pVPakc Vectors, pCMV vectors, pSG5 vectors(Stratagene)), retroviral vectors (e.g., pFB vectors (Stratagene)),pCDNA-3 (Invitrogen) or modified forms thereof, adenoviral vectors;Adeno-associated virus vectors, baculovirus vectors, yeast vectors(e.g., pESC vectors (Stratagene)).

Nucleic acid sequences encoding CTLA4 mutant molecules can integrateinto the genome of the eukaryotic host cell and replicate as the hostgenome replicates. Alternatively, the vector carrying CTLA4 mutantmolecules can contain origins of replication allowing forextrachromosomal replication.

For expressing the nucleic acid sequences in Saccharomyces cerevisiae,the origin of replication from the endogenous yeast plasmid, the 2μcircle can be used. (Broach, (1983) Meth. Enz. 101:307). Alternatively,sequences from the yeast genome capable of promoting autonomousreplication can be used (see, for example, Stinchcomb et al., (1979)Nature 282:39); Tschemper et al., (1980) Gene 10:157; and Clarke et al.,(1983) Meth. Enz. 101:300).

Transcriptional control sequences for yeast vectors include promotersfor the synthesis of glycolytic enzymes (Hess et al., (1968) J. Adv.Enzyme Reg. 7:149; Holland et al., (1978) Biochemistry 17:4900).Additional promoters known in the art include the CMV promoter providedin the CDM8 vector (Toyama and Okayama, (1990) FEBS 268:217-221); thepromoter for 3-phosphoglycerate kinase (Hitzeman et al., (1980) J. Biol.Chem. 255:2073), and those for other glycolytic enzymes.

Other promoters are inducible because they can be regulated byenvironmental stimuli or the growth medium of the cells. These induciblepromoters include those from the genes for heat shock proteins, alcoholdehydrogenase 2, isocytochrome C, acid phosphatase, enzymes associatedwith nitrogen catabolism, and enzymes responsible for maltose andgalactose utilization.

Regulatory sequences may also be placed at the 3′ end of the codingsequences. These sequences may act to stabilize messenger RNA. Suchterminators are found in the 3′ untranslated region following the codingsequences in several yeast-derived and mammalian genes.

Exemplary vectors for plants and plant cells include, but are notlimited to, Agrobacterium T_(i) plasmids, cauliflower mosaic virus(CaMV), and tomato golden mosaic virus (TGMV).

General aspects of mammalian cell host system transformations have beendescribed by Axel (U.S. Pat. No. 4,399,216 issued Aug. 16, 1983).Mammalian cells can be transformed by methods including but not limitedto, transfection in the presence of calcium phosphate, microinjection,electroporation, or via transduction with viral vectors.

Methods for introducing foreign DNA sequences into plant and yeastgenomes include (1) mechanical methods, such as microinjection of DNAinto single cells or protoplasts, vortexing cells with glass beads inthe presence of DNA, or shooting DNA-coated tungsten or gold spheresinto cells or protoplasts; (2) introducing DNA by making cell membranespermeable to macromolecules through polyethylene glycol treatment orsubjection to high voltage electrical pulses (electroporation); or (3)the use of liposomes (containing cDNA) which fuse to cell membranes.

Expression of CTLA4 mutant molecules can be detected by methods known inthe art. For example, the mutant molecules can be detected by Coomassiestaining SDS-PAGE gels and immunoblotting using antibodies that bindCTLA4. Protein recovery can be performed using standard proteinpurification means, e.g., affinity chromatography or ion-exchangechromatography, to yield substantially pure product (R. Scopes in:“Protein Purification, Principles and Practice”, Third Edition,Springer-Verlag (1994)).

The invention further provides soluble CTLA4 mutant molecules producedabove herein.

CTLA4Ig Codon-Based Mutagenesis

In one embodiment, site-directed mutagenesis and a novel screeningprocedure were used to identify several mutations in the extracellulardomain of CTLA4 that improve binding avidity for CD86. In thisembodiment, mutations were carried out in residues in the regions of theextracellular domain of CTLA4 from serine 25 to arginine 33, the C′strand (alanine 49 and threonine 51), the F strand (lysine 93, glutamicacid 95 and leucine 96), and in the region from methionine 97 throughtyrosine 102, tyrosine 103 through glycine 107 and in the G strand atpositions glutamine 111, tyrosine 113 and isoleucine 115. These siteswere chosen based on studies of chimeric CD28/CTLA4 fusion proteins(Peach et al., J. Exp. Med., 1994, 180:2049-2058), and on a modelpredicting which amino acid residue side chains would be solventexposed, and a lack of amino acid residue identity or homology atcertain positions between CD28 and CTLA4. Also, any residue which isspatially in close proximity (5 to 20 Angstrom Units) to the identifiedresidues is considered part of the present invention.

To synthesize and screen soluble CTLA4 mutant molecules with alteredaffinities for CD80 and/or CD86, a two-step strategy was adopted. Theexperiments entailed first generating a library of mutations at aspecific codon of an extracellular portion of CTLA4 and then screeningthese by BIAcore analysis to identify mutants with altered reactivity toCD80 or CD86. The Biacore assay system (Pharmacia, Piscataway, N.J.)uses a surface plasmon resonance detector system that essentiallyinvolves covalent binding of either CD80Ig or CD86Ig to a dextran-coatedsensor chip which is located in a detector. The test molecule can thenbe injected into the chamber containing the sensor chip and the amountof complementary protein that binds can be assessed based on the changein molecular mass which is physically associated with the dextran-coatedside of the sensor chip; the change in molecular mass can be measured bythe detector system.

Advantages of the Invention

Because CTLA4 binding to CD80 and CD86 is characterized by rapid “on”rates and rapid dissociation (“off”) rates, and because CTLA4Ig-CD86complexes dissociate approximately 5- to 8-fold more rapidly thanCTLA4Ig-CD80 complexes, it was reasoned that slowing the rate ofdissociation of CTLA4Ig from CD80 and/or CD86 would result in moleculeswith more potent immunosuppressive properties. Thus, soluble CTLA4mutant molecules having a higher avidity for CD80- or CD86-positivecells compared to wild type CTLA4, or non-mutated forms of CTLA4Ig, areexpected to block the priming of antigen specific activated cells withhigher efficiency than wild type CTLA4 or non-mutated forms of CTLA4Ig.

Further, production costs for CTLA4Ig are very high. The high aviditymutant CTLA4Ig molecules having higher potent immunosuppressiveproperties can be used in the clinic, at considerably lower doses thannon-mutated CTLA4Ig, to achieve similar levels of immunosuppression.Thus, soluble CTLA4 mutant molecules, e.g., L104EA29YIg, may be verycost effective.

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

EXAMPLES Example 1

This example provides a description of the methods used to generate thenucleotide sequences encoding the soluble CTLA4 mutant molecules of theinvention. A single-site mutant L104EIg was generated and tested forbinding kinetics for CD80 and/or CD86. The L104EIg nucleotide sequencewas used as a template to generate the double-site mutant CTLA4sequence, L104EA29YIg, which was tested for binding kinetics for CD80and/or CD86.

CTLA4Ig Codon Based Mutagenesis

A mutagenesis and screening strategy was developed to identify mutantCTLA4Ig molecules that had slower rates of dissociation (“off” rates)from CD80 and/or CD86 molecules. Single-site mutant nucleotide sequenceswere generated using CTLA4Ig (U.S. Pat. Nos. 5,844,095; 5,851,795; and5,885,796; ATCC Accession No. 68629) as a template. Mutagenicoligonucleotide PCR primers were designed for random mutagenesis of aspecific cDNA codon by allowing any base at positions 1 and 2 of thecodon, but only guanine or thymine at position 3 (XXG/T; also known asNNG/T). In this manner, a specific codon encoding an amino acid could berandomly mutated to code for each of the 20 amino acids. In that regard,XXG/T mutagenesis yields 32 potential codons encoding each of the 20amino acids. PCR products encoding mutations in close proximity to−M97-G107 of CTLA4Ig (see FIG. 7 or 8), were digested with SacI/XbaI andsubcloned into similarly cut CTLA4Ig πLN (also known as piLN) expressionvector. This method was used to generate the single-site CTLA4 mutantmolecule L104EIg (FIG. 8).

For mutagenesis in proximity to S25-R33 of CTLA4Ig, a silent NheIrestriction site was first introduced 5′ to this loop, by PCRprimer-directed mutagenesis. PCR products were digested with NheI/XbaIand subcloned into similarly cut CTLA4Ig or L104EIg expression vectors.This method was used to generate the double-site CTLA4 mutant moleculeL104EA29YIg (FIG. 7). In particular, the nucleic acid molecule encodingthe single-site CTLA4 mutant molecule, L104EIg, was used as a templateto generate the double-site CTLA4 mutant molecule, L104EA29YIg. The piLNvector having the L104EA29YIg is shown in FIG. 12.

Example 2

The following provides a description of the screening methods used toidentify the single- and double-site mutant CTLA4 polypeptides,expressed from the constructs described in Example 1, that exhibited ahigher binding avidity for CD80 and CD86 antigens, compared tonon-mutated CTLA4Ig molecules.

Current in vitro and in vivo studies indicate that CTLA4Ig by itself isunable to completely block the priming of antigen specific activated Tcells. In vitro studies with CTLA4Ig and either monoclonal antibodyspecific for CD80 or CD86 measuring inhibition of T cell proliferationindicate that anti-CD80 monoclonal antibody did not augment CTLA4Iginhibition. However, anti-CD86 monoclonal antibody did augment theinhibition, indicating that CTLA4Ig was not as effective at blockingCD86 interactions. These data support earlier findings by Linsley et al.(Immunity, (1994), 1:793-801) showing inhibition of CD80-mediatedcellular responses required approximately 100 fold lower CTLA4Igconcentrations than for CD86-mediated responses. Based on thesefindings, it was surmised that soluble CTLA4 mutant molecules having ahigher avidity for CD86 than wild type CTLA4 should be better able toblock the priming of antigen specific activated cells than CTLA4Ig.

To this end, the soluble CTLA4 mutant molecules described in Example 1above were screened using a novel screening procedure to identifyseveral mutations in the extracellular domain of CTLA4 that improvebinding avidity for CD80 and CD86. This screening strategy provided aneffective method to directly identify mutants with apparently slower“off” rates without the need for protein purification or quantitationsince “off” rate determination is concentration independent (O'Shannessyet al., (1993) Anal. Biochem., 212:457-468).

COS cells were transfected with individual miniprep purified plasmid DNAand propagated for several days. Three day conditioned culture media wasapplied to BIAcore biosensor chips (Pharmacia Biotech AB, Uppsala,Sweden) coated with soluble CD80Ig or CD86Ig. The specific binding anddissociation of mutant proteins was measured by surface plasmonresonance (O'Shannessy, D. J., et al., (1993) Anal. Biochem.212:457-468). All experiments were run on BIAcore™ or BIAcore™ 2000biosensors at 25° C. Ligands were immobilized on research grade NCMSsensor chips (Pharmacia) using standard N-ethyl-N′-(dimethylaminopropyl)carbodiimidN-hydroxysuccinimide coupling (Johnsson, B., et al. (1991)Anal. Biochem. 198: 268-277; Khilko, S. N., et al. (1993) J. Biol. Chem.268:5425-15434).

Screening Method

COS cells grown in 24 well tissue culture plates were transientlytransfected with DNA encoding mutant CTLA4Ig. Culture media containingsecreted soluble mutant CTLA4Ig was collected 3 days later.

Conditioned COS cell culture media was allowed to flow over BIAcorebiosensor chips derivatized with CD86Ig or CD80Ig (as described inGreene et al., 1996 J. Biol. Chem. 271:26762-26771), and mutantmolecules were identified with “off” rates slower than that observed forwild type CTLA4Ig. The cDNAs corresponding to selected media sampleswere sequenced and DNA was prepared to perform larger scale COS celltransient transfection, from which mutant CTLA4Ig protein was preparedfollowing protein A purification of culture media.

BIAcore analysis conditions and equilibrium binding data analysis wereperformed as described in J. Greene et al. 1996 J. Biol. Chem.271:26762-26771, and as described herein.

BIAcore Data Analysis

Senosorgram baselines were normalized to zero response units (RU) priorto analysis. Samples were run over mock-derivatized flow cells todetermine background response unit (RU) values due to bulk refractiveindex differences between solutions. Equilibrium dissociation constants(K_(d)) were calculated from plots of R_(eq) versus C, where R_(eq) isthe steady-state response minus the response on a mock-derivatized chip,and C is the molar concentration of analyte. Binding curves wereanalyzed using commercial nonlinear curve-fitting software (Prism,GraphPAD Software).

Experimental data were first fit to a model for a single ligand bindingto a single receptor (1-site model, i.e., a simple langmuir system,A+B↔AB), and equilibrium association constants (K_(d)=[A]·[B]\[AB]) werecalculated from the equation R=R_(max)·C/(K_(d)+C). Subsequently, datawere fit to the simplest two-site model of ligand binding (i.e., to areceptor having two non-interacting independent binding sites asdescribed by the equationR=R_(max1)·C\(K_(d1)+C)+R_(max2)·C\(K_(d2)+C)).

The goodness-of-fits of these two models were analyzed visually bycomparison with experimental data and statistically by an F test of thesums-of-squares. The simpler one-site model was chosen as the best fit,unless the two-site model fit significantly better (p<0.1).

Association and disassociation analyses were performed using BIAevaluation 2.1 Software (Pharmacia). Association rate constants k_(on)were calculated in two ways, assuming both homogenous single-siteinteractions and parallel two-site interactions. For single-siteinteractions, k_(on) values were calculated according to the equationR_(t)=R_(eq)(1−exp^(−ks(t-t) ₀) where R_(t) is a response at a giventime, t; R_(eq) is the steady-state response; t₀ is the time at thestart of the injection; and k_(s)=dR/dt=k_(on)·Ck_(off), and where C isa concentration of analyte, calculated in terms of monomeric bindingsites. For two-site interactions k_(on) values were calculated accordingto the equation R_(t)=R_(eq1)(1−exp^(−ks1(t-t) ₀⁾+R_(eq2)(1−exp^(ks2(t-t) ₀). For each model, the values of k_(on) weredetermined from the calculated slope (to about 70% maximal association)of plots of k_(s) versus C.

Dissociation data were analyzed according to one site (AB=A+B) or twosites (AiBj=Ai+Bj) models, and rate constants (k_(off)) were calculatedfrom best fit curves. The binding site model was used except when theresiduals were greater than machine background (2-10 RU, according tomachine), in which case the two-binding site model was employed.Half-times of receptor occupancy were calculated using the relationshipt_(1/2)=0.693/k_(off).

Flow Cytometry

Murine mAb L307.4 (anti-CD80) was purchased from Becton Dickinson (SanJose, Calif.) and IT2.2 (anti-B7-0 [also known as CD86]), fromPharmingen (San Diego, Calif.). For immunostaining, CD80-positive and/orCD86-positive CHO cells were removed from their culture vessels byincubation in phosphate-buffered saline (PBS) containing 10 mM EDTA. CHOcells (1-10×10⁵) were first incubated with mAbs or immunoglobulin fusionproteins in DMEM containing 10% fetal bovine serum (FBS), then washedand incubated with fluorescein isothiocyanate-conjugated goat anti-mouseor anti-human immunoglobulin second step reagents (Tago, Burlingame,Calif.). Cells were given a final wash and analyzed on a FACScan (BectonDickinson).

SDS-PAGE and Size Exclusion Chromatography

SDS-PAGE was performed on Tris/glycine 4-20% acrylamide gels (Novex, SanDiego, Calif.). Analytical gels were stained with Coomassie Blue, andimages of wet gels were obtained by digital scanning. CTLA4Ig (25 μg)and L104EA29YIg (25 μg) were analyzed by size exclusion chromatographyusing a TSK-GEL G300 SW_(XL) column (7.8×300 mm, Tosohaas,Montgomeryville, Pa.) equilibrated in phosphate buffered salinecontaining 0.02% NAN₃ at a flow rate of 1.0 ml/min.

CTLA4X_(C120S) and L104EA29YX_(C120S).

Single chain CTLA4X_(X120S) was prepared as previously described(Linsley et al., (1995) J. Biol. Chem., 270:15417-15424). Briefly, anoncostatin M CTLA4 (OMCTLA4) expression plasmid was used as a template,the forward primer,

GAGGTGATAAAGCTTCACCAATGGGTGTACTGCTCACACAGwas chosen to match sequences in the vector; and the reverse primer,

GTGGTGTATTGGTCTAGATCAATCAGAATCTGGGCACGGTTCcorresponded to the last seven amino acids (i.e. amino acids 118-124) inthe extracellular domain of CTLA4, and contained a restriction enzymesite, and a stop codon (TGA). The reverse primer specified a C120S(cysteine to serine at position 120) mutation. In particular, thenucleotide sequence GCA (nucleotides 34-36) of the reverse primer shownabove is replaced with one of the following nucleotide sequences: AGA,GGA, TGA, CGA, ACT, or GCT. As persons skilled in the art willunderstand, the nucleotide sequence GCA is a reversed complementarysequence of the codon TGC for cysteine. Similarly, the nucleotidesequences AGA, GGA, TGA, CGA, ACT, or GCT are the reversed complementarysequences of the codons for serine. Polymerase chain reaction productswere digested with HindIII/XbaI and directionally subcloned into theexpression vector πLN (Bristol-Myers Squibb Company, Princeton, N.J.).L104EA29YX_(C120S) was prepared in an identical manner. Each constructwas verified by DNA sequencing.

Identification and Biochemical Characterization of High Avidity Mutants

Twenty four amino acids were chosen for mutagenesis and the resulting2300 mutant proteins assayed for CD86Ig binding by surface plasmonresonance (SPR; as described, supra). The predominant effects ofmutagenesis at each site are summarized in Table II. Random mutagenesisof some amino acids in the S25-R33 apparently did not alter ligandbinding. Mutagenesis of E31 and R33 and residues M97-Y102 apparentlyresulted in reduced ligand binding. Mutagenesis of residues, S25, A29,and T30, K93, L96, Y103, L104, and G105, resulted in proteins with slow“on” and/or slow “off” rates. These results confirm previous findingsthat residues in the S25-R33 region, and residues in or near M97-Y102influence ligand binding (Peach et al., (1994) J. Exp. Med.,180:2049-2058.

Mutagenesis of sites S25, T30, K93, L96, Y103, and G105 resulted in theidentification of some mutant proteins that had slower “off” rates fromCD86Ig. However, in these instances, the slow “off” rate was compromisedby a slow “on” rate which resulted in mutant proteins with an overallavidity for CD86Ig that was apparently similar to that seen with wildtype CTLA4Ig. In addition, mutagenesis of K93 resulted in significantaggregation which may have been responsible for the kinetic changesobserved.

Random mutagenesis of L104 followed by COS cell transfection andscreening by SPR of culture media samples over immobilized CD86Igyielded six media samples containing mutant proteins with approximately2-fold slower “off” rates than wild type CTLA4Ig. When the correspondingcDNA of these mutants were sequenced, each was found to encode a leucineto glutamic acid mutation (L104E). Apparently, substitution of leucine104 to aspartic acid (L104D) did not affect CD86Ig binding.

Mutagenesis was then repeated at each site listed in Table II, this timeusing L104E as the PCR template instead of wild type CTLA4Ig, asdescribed above. SPR analysis, again using immobilized CD86Ig,identified six culture media samples from mutagenesis of alanine 29 withproteins having approximately 4-fold slower “off” rates than wild typeCTLA4Ig. The two slowest were tyrosine substitutions (L104EA29Y), twowere leucine (L104EA29L), one was tryptophan (L104EA29W), and one wasthreonine (L104EA29T). Apparently, no slow “off” rate mutants wereidentified when alanine 29 was randomly mutated, alone, in wild typeCTLA4Ig.

The relative molecular mass and state of aggregation of purified L104Eand L104EA29YIg was assessed by SDS-PAGE and size exclusionchromatography. L104EA29YIg (˜1 μg; lane 3) and L104EIg (˜1 μg; lane 2)apparently had the same electrophoretic mobility as CTLA4Ig (˜1 μg;lane 1) under reducing (˜50 kDa; +ßME; plus 2-mercaptoethanol) andnon-reducing (˜100 kDa; −ßME) conditions (FIG. 10A). Size exclusionchromatography demonstrated that L104EA29YIg (FIG. 10C) apparently hadthe same mobility as dimeric CTLA4Ig (FIG. 10B). The major peaksrepresent protein dimer while the faster eluting minor peak in FIG. 10Brepresents higher molecular weight aggregates. Approximately 5.0% ofCTLA4Ig was present as higher molecular weight aggregates but there wasno evidence of aggregation of L104EA29YIg or L104EIg. Therefore, thestronger binding to CD86Ig seen with L104EIg and L104EA29YIg could notbe attributed to aggregation induced by mutagenesis.

Equilibrium and Kinetic Binding Analysis

Equilibrium and kinetic binding analysis was performed on protein Apurified CTLA4Ig, L104EIg, and L104EA29YIg using surface plasmonresonance (SPR). The results are shown in Table I. Observed equilibriumdissociation constants (K_(d); Table I) were calculated from bindingcurves generated over a range of concentrations (5.0-200 nM).L104EA29YIg binds more strongly to CD86Ig than does L104EIg or CTLA4Ig.The lower K_(d) of L104EA29YIg (3.21 nM) than L104EIg (6.06 nM) orCTLA4Ig (13.9 nM) indicates higher binding avidity of L104EA29YIg toCD86Ig. The lower K_(d) of L104EA29YIg (3.66 nM) than L104EIg (4.47 nM)or CTLA4Ig (6.51 nM) indicates higher binding avidity of L104EA29YIg toCD80Ig.

Kinetic binding analysis revealed that the comparative “on” rates forCTLA4Ig, L104EIg, and L104EA29YIg binding to CD80 were similar, as werethe “on” rates for CD86Ig (Table I). However, “off” rates for thesemolecules were not equivalent (Table I). Compared to CTLA4Ig,L104EA29YIg had approximately 2-fold slower “off” rate from CD80Ig, andapproximately 4-fold slower “off” rate from CD86Ig. L104E had “off”rates intermediate between L104EA29YIg and CTLA4Ig. Since theintroduction of these mutations did not significantly affect “on” rates,the increase in avidity for CD80Ig and CD86Ig observed with L104EA29YIgwas likely primarily due to a decrease in “off” rates.

To determine whether the increase in avidity of L104EA29YIg for CD86Igand CD80Ig was due to the mutations affecting the way each monomerassociated as a dimer, or whether there were avidity enhancingstructural changes introduced into each monomer, single chain constructsof CTLA4 and L104EA29Y extracellular domains were prepared followingmutagenesis of cysteine 120 to serine as described supra, and by Linsleyet al., (1995) J Biol. Chem., 270:15417-15424. The purified proteinsCTLA4X_(C120S) and L104EA29YX_(C120S) were shown to be monomeric by gelpermeation chromatography (Linsley et al., (1995), supra), before theirligand binding properties were analyzed by SPR. Results showed thatbinding affinity of both monomeric proteins for CD86Ig was approximately35-80-fold less than that seen for their respective dimers (Table I).This supports previously published data establishing that dimerizationof CTLA4 was required for high avidity ligand binding (Greene et al.,(1996) J. Biol. Chem., 271:26762-26771).

L104EA29YX_(C120S) bound with approximately 2-fold higher affinity thanCTLA4X_(C120S) to both CD80Ig and CD86Ig. The increased affinity was dueto approximately 3-fold slower rate of dissociation from both ligands.Therefore, stronger ligand binding by L104EA29Y was most likely due toavidity enhancing structural changes that had been introduced into eachmonomeric chain rather than alterations in which the molecule dimerized.

Location and Structural Analysis of Avidity Enhancing Mutations

The solution structure of the extracellular IgV-like domain of CTLA4 hasrecently been determined by NMR spectroscopy (Metzler et al., (1997)Nature Struct. Biol., 4:527-531. This allowed accurate location ofleucine 104 and alanine 29 in the three dimensional fold (FIG. 11A-B).Leucine 104 is situated near the highly conserved MYPPPY amino acidsequence (SEQ ID NO:9). Alanine 29 is situated near the C-terminal endof the S25-R33 region, which is spatially adjacent to the MYPPPY region(SEQ ID NO:9). While there is significant interaction between residuesat the base of these two regions, there is apparently no directinteraction between L104 and A29 although they both comprise part of acontiguous hydrophobic core in the protein. The structural consequencesof the two avidity enhancing mutants were assessed by modeling. The A29Ymutation can be easily accommodated in the cleft between the S25-R33region and the MYPPPY region (SEQ ID NO:9), and may serve to stabilizethe conformation of the MYPPPY region (SEQ ID NO:9). In wild type CTLA4,L104 forms extensive hydrophobic interactions with L96 and V94 near theMYPPPY region (SEQ ID NO:9). It is highly unlikely that the glutamicacid mutation adopts a conformation similar to that of L104 for tworeasons. First, there is insufficient space to accommodate the longerglutamic acid side chain in the structure without significantperturbation to the S25-R33 region. Second, the energetic costs ofburying the negative charge of the glutamic acid side chain in thehydrophobic region would be large. Instead, modeling studies predictthat the glutamic acid side chain flips out on to the surface where itscharge can be stabilized by solvation. Such a conformational change caneasily be accommodated by G105, with minimal distortion to otherresidues in the regions.

Binding of High Avidity Mutants to CHO Cells Expressing CD80 or CD86

FACS analysis (FIG. 2) of CTLA4Ig and mutant molecules binding to stablytransfected CD80+ and CD86+CHO cells was performed as described herein.CD80-positive and CD86-positive CHO cells were incubated with increasingconcentrations of CTLA4Ig, L104EA29YIg, or L104EIg, and then washed.Bound immunoglobulin fusion protein was detected using fluoresceinisothiocyanate-conjugated goat anti-human immunoglobulin.

As shown in FIG. 2, CD80-positive or CD86-positive CHO cells (1.5×10⁵)were incubated with the indicated concentrations of CTLA4Ig (closedsquares), L104EA29YIg (circles), or L104EIg (triangles) for 2 hr. at 23°C., washed, and incubated with fluorescein isothiocyanate-conjugatedgoat anti-human immunoglobulin antibody. Binding on a total of 5,000viable cells was analyzed (single determination) on a FACScan, and meanfluorescence intensity (MFI) was determined from data histograms usingPC-LYSYS. Data were corrected for background fluorescence measured oncells incubated with second step reagent only (MFI=7). Control L6 mAb(80 μg/ml) gave MFI<30. These results are representative of fourindependent experiments.

Binding of L104EA29YIg, L104EIg, and CTLA4Ig to human CD80-transfectedCHO cells is approximately equivalent (FIG. 2A). L104EA29YIg and L104EIgbind more strongly to CHO cells stably transfected with human CD86 thandoes CTLA4Ig (FIG. 2B).

Functional Assays

Human CD4-positive T cells were isolated by immunomagnetic negativeselection (Linsley et al., (1992) J Exp. Med. 176:1595-1604). IsolatedCD4-positive T cells were stimulated with phorbal myristate acetate(PMA) plus CD80-positive or CD86-positive CHO cells in the presence oftitrating concentrations of inhibitor. CD4-positive T cells(8-10×10⁴/well) were cultured in the presence of 1 nM PMA with orwithout irradiated CHO cell stimulators. Proliferative responses weremeasured by the addition of 1 μCi/well of [3H]thymidine during the final7 hours of a 72 hour culture. Inhibition of PMA plus CD80-positive CHO,or CD86-positive CHO, stimulated T cells by L104EA29YIg and CTLA4Ig wasperformed. The results are shown in FIG. 3. L104EA29YIg inhibitsproliferation of CD80-positive PMA treated CHO cells more than CTLA4Ig(FIG. 3A). L104EA29YIg is also more effective than CTLA4Ig at inhibitingproliferation of CD86-positive PMA treated CHO cells (FIG. 3B).Therefore, L104EA29YIg is a more potent inhibitor of both CD80- andCD86-mediated costimulation of T cells.

FIG. 4 shows inhibition by L104EA29YIg and CTLA4Ig of allostimulatedhuman T cells prepared above, and further allostimulated with a human Blymphoblastoid cell line (LCL) called PM that expressed CD80 and CD86 (Tcells at 3.0×10⁴/well and PM at 8.0×10³/well). Primary allostimulationoccurred for 6 days, then the cells were pulsed with ³H-thymidine for 7hours, before incorporation of radiolabel was determined.

Secondary allostimulation was performed as follows. Seven day primaryallostimulated T cells were harvested over lymphocyte separation medium(LSM) (ICN, Aurora, Ohio) and rested for 24 hours. T cells were thenrestimulated (secondary), in the presence of titrating amounts ofCTLA4Ig or L104EA29YIg, by adding PM in the same ratio as above.Stimulation occurred for 3 days, then the cells were pulsed withradiolabel and harvested as above. The effect of L104EA29YIg on primaryallostimulated T cells is shown in FIG. 4A. The effect of L104EA29YIg onsecondary allostimulated T cells is shown in FIG. 4B. L104EA29YIginhibits both primary and secondary T cell proliferative responsesbetter than CTLA4Ig.

To measure cytokine production (FIG. 5), duplicate secondaryallostimulation plates were set up. After 3 days, culture media wasassayed using ELISA kits (Biosource, Camarillo, Calif.) using conditionsrecommended by the manufacturer. L104EA29YIg was found to be more potentthan CTLA4Ig at blocking T cell IL-2, IL-4, and γ-IFN cytokineproduction following a secondary allogeneic stimulus (FIGS. 5A-C).

The effects of L104EA29YIg and CTLA4Ig on monkey mixed lymphocyteresponse (MLR) are shown in FIG. 6. Peripheral blood mononuclear cells(PBMC'S; 3.5×10⁴ cells/well from each monkey) from 2 monkeys werepurified over lymphocyte separation medium (LSM) and mixed with 2 μg/mlphytohemaglutinin (PHA). The cells were stimulated 3 days then pulsedwith radiolabel 16 hours before harvesting. L104EA29YIg inhibited monkeyT cell proliferation better than CTLA4Ig.

TABLE I Equilibrium and apparent kinetic constants are given in thefollowing table (values are means ± standard deviation from threedifferent experiments): Immobilized k_(on) (×10⁵) k_(off) (×10⁻³) K_(d)Protein Analyte M⁻¹ S⁻¹ S⁻¹ nM CD80Ig CTLA4Ig 3.44 ± 0.29 2.21 ± 0.186.51 ± 1.08 CD80Ig L104EIg 3.02 ± 0.05 1.35 ± 0.08 4.47 ± 0.36 CD80IgL104EA29YIg 2.96 ± 0.20 1.08 ± 0.05 3.66 ± 0.41 CD80Ig CTLA4X_(C120S)12.0 ± 1.0  230 ± 10  195 ± 25  CD80Ig L104EA29YX_(C120S)  8.3 ± 0.26 71± 5  85.0 ± 2.5  CD86Ig CTLA4Ig 5.95 ± 0.57 8.16 ± 0.52 13.9 ± 2.27CD86Ig L104EIg 7.03 ± 0.22 4.26 ± 0.11 6.06 ± 0.05 CD86Ig L104EA29YIg6.42 ± 0.40 2.06 ± 0.03 3.21 ± 0.23 CD86Ig CTLA4X_(C120S) 16.5 ± 0.5 840 ± 55  511 ± 17  CD86Ig L104EA29YX_(C120S) 11.4 ± 1.6  300 ± 10  267± 29 

TABLE II The effect on CD86Ig binding by mutagenesis of CTLA4Ig at thesites listed was determined by SPR, described supra. The predominanteffect is indicated with a “+” sign. Effects of Mutagenesis Slow “on”Reduced Mutagenesis No Apparent rate/slow ligand Site Effect “off ratebinding S25 + P26 + G27 + K28 + A29 + T30 + E31 + R33 + K93 + L96 +M97 + Y98 + P99 + P100 + P101 + Y102 + Y103 + L104 + G105 + I106 +G107 + Q111 + Y113 + I115 +

As will be apparent to those skilled in the art to which the inventionpertains, the present invention may be embodied in forms other thanthose specifically disclosed above without departing from the spirit oressential characteristics of the invention. The particular embodimentsof the invention described above, are, therefore, to be considered asillustrative and not restrictive. The scope of the present invention isas set forth in the appended claims rather than being limited to theexamples contained in the foregoing description.

What is claimed is:
 1. A method for inhibiting graft versus host diseasein a subject which comprises administering to the subject a solubleCTLA4 molecule comprising an extracellular domain as shown in SEQ IDNO:8 beginning with alanine at position 26 or methionine at position 27and ending with aspartic acid at position
 150. 2. The method of claim 1wherein the soluble CTLA4 molecule further comprising an amino acidsequence which alters the solubility, affinity or valency of the solubleCTLA4 mutant molecule.
 3. The method of claim 2, wherein the solubleCTLA4 molecule comprises a human immunoglobulin constant region.
 4. Themethod of claim 3 wherein the immunoglobulin constant region is mutatedto reduce effector function.
 5. The method of claim 3 wherein theimmunoglobulin constant region comprises a hinge, CH2 and CH3 regions ofan immunoglobulin molecule.
 6. The method of claim 5, wherein any or allof the cysteine residues within the immunoglobulin hinge are substitutedwith serine.
 7. The method of claim 6 wherein a cysteine at position 156is substituted with a serine, a cysteine at position 162 is substitutedwith a serine, and a cysteine at position 165 is substituted with aserine, as shown in SEQ ID NO:8.
 8. The method of claim 1, wherein thesoluble CTLA4 molecule further comprising a junction amino acid residueand an immunoglobulin, where the junction amino acid residue is locatedbetween the amino acid sequence which ends with aspartic acid atposition 150 and the immunoglobulin.
 9. The method of claim 8, whereinthe junction amino acid residue is glutamine.
 10. A method forinhibiting graft versus host disease in a subject which comprisesadministering to the subject a soluble CTLA4-Ig molecule comprising theamino acid sequence shown in SEQ ID NO:8 beginning with alanine atposition 26 or methionine at position 27 and ending with lysine atposition 383.